Serum thyroid autoantibodies in malignant thyroid nodules

Background: Thyroid cancer is one of the most common malignant tumours of the endocrine system. Thyroid autoantibodies were reported to be associated with malignant thyroid nodules. This study aimed to assess the diagnostic accuracy of serum thyroid autoantibody levels for malignant thyroid nodules. Methods: From March 2023 to January 2024, we recruited 104 consecutive patients with thyroid nodules confirmed by ultra-sonography in three departments of Bangabandhu Sheikh Mujib Medical University. Out of these, 52 were diagnosed with malignant thyroid nodules using fine needle aspiration cytology, while the remaining 52 had benign thyroid nodules. Serum thyroid autoantibodies were measured using the immunoassay technique. Results: The mean levels of serum thyroid peroxidase antibody (TPOAb) were significantly higher ( P <0.001) in the malignant group compared to the benign group (36.1 versus 24.0 IU/mL). The mean levels of serum thyroglobulin antibodies (TgAb) were also significantly higher ( P <0.001) in the malignant group (39.8 IU/mL versus 29.1 IU/mL). Elevated TPOAb (>40 IU/mL) and TgAb (>35 IU/mL) showed reasonable accuracy (60% to 65%) in detecting malignancy of thyroid nodules. Conclusions: Thyroid malignancy is positively associated with TgAb and TPOAb. Therefore, thyroid autoantibodies could be considered for screening malignancy in thyroid nodules.


INTRODUCTION
The American Thyroid Association has defined thyroid nodules as discrete lesions within the thyroid gland, radiologically distinct from surrounding thyroid parenchyma. 1 The prevalence of thyroid nodules in the general population increases from 8% to 76% with highresolution ultrasound instead of clinical examination. 2e incidence of thyroid cancer has been increasing rapidly in recent years, with approximately 14.3 cases per 100,000 per year. 3Its incidence is 43.8 cases per 100,000 person-years among the East Asian people. 4In India, the incidence of thyroid cancer is 5.8 per 100,000 people. 5The incidence has increased worldwide in the last three decades. 6yroglobulin antibody (TgAb), an immunoglobulin G glycoprotein secreted from lymphoid B cells, becomes hyperfunctioning during malignancy. 7hyroid peroxidase antibodies (TPOAb) usually indicate autoimmune thyroid disease when they are positive. 8f autoimmune thyroid disease, especially in young patients. 10Measuring thyroid autoantibodies such as TPOAb and TgAb is a cost-effective and reliable test.
Thus, this study aimed to assess the diagnostic accuracy of serum thyroid autoantibody levels for malignant thyroid nodules.

Study design
This cross-sectional study was done from  11 were excluded from the study.

Sample size determination
Considering the formula 12 Where n (number of cases); u=1.96 for 1% level of significance; v=0.52 (from z table) at 70% power of the test; π₀ (mean in group I)=44; π1 (mean in group II) = 27; πbar = (π₀ +π1)/2=35.Therefore, the sample size for each group was 52, and the total sample size was 104.

Specimen collection
Venous blood (3 mL) samples were collected in a biochemistry tube from the antecubital vein.After an incubation period of 30 minutes, the biochemistry tube was centrifuged at 3,000 revolutions per minute for 5 With all aseptic precautions, a 10 ml syringe was attached to a syringe holder.Two fingers of the free hand firmly grasped the nodule while the other hand held a pistol grip syringe holder.The needle was then inserted into the nodule through the skin.Once the needle tip was in the nodule, gentle suction was applied.
The needle was moved in and out vertically within the nodule.This allowed the cellular material to dislodge and easily aspirate into the needle.Suction was maintained, and as soon as aspirate appeared in the half of the needle, the suction was released, and the needle was withdrawn.The needle was reattached to the syringe with the beveled end facing down, and one drop of aspirate material was ejected onto several glass slides.All the slides were labelled.

Measurement of thyroid autoantibodies
TgAb and TPOAb were measured using the immunoassay technique (ADVIA centaur XPT, Siemens) in the Department of Microbiology and Immunology, BSMMU.

Statistical analysis
Data were entered, cleaned, and analysed using SPSS version 26.Quantitative data were described using mean and standard deviation, and compared using the t Serum thyroid autoantibodies in malignant thyroid nodules 2 of 5

HIGHLIGHTS
1. Limited research exists on thyroid antibodies in malignant thyroid tumours.
3. These two antibodies can be used to screen for malignancy of thyroid nodules.
test.The results of TgAb and TPOAb were considered elevated if these were >40 IU/mL 13 and >35 IU/mL 14 , respectively.Qualitative data were described using frequency and percent and association was measured using chi-square test.Logistic regression analysis was done to examine the associated factors for malignant forwards.Sensitivity, specificity, predictive values and diagnostic accuracy 15 were calculated.P<0.05 was considered as significant.

Ethical issues
Following the ethical approval, we have explained the potential risk of the FNAC procedure to the participants.Informed written consent was taken from the participants.Confidentiality of the personal information was maintained.

RESULTS
The mean age of the patients with malignant (43.9 years) and benign (42.4 years) nodules were similar.
The mean serum TgAb levels are higher in the malignant group (39.8 IU/mL) compared to the benign group (29.1 IU/mL).TPOAb levels exhibited higher in the malignant group (36.1 IU/mL) compared to the benign group (24.0 IU/mL) (TABLE 1).These differences persisted even after adjustment of the potential confounders using a multivariate logistic regression analysis (data not shown).Elevated levels of TgAb were significantly associated with thyroid malignancy (OR 2.6; 95% CI 1.1, 5.9).This was true for TPOAb (OR 4.0; 95% CI 1.7, 9.5) also (TABLE 2).

DISCUSSION
There is a dearth of information on the usefulness of thyroid autoantibody levels in diagnosing malignant thyroid nodules.We have examined this relationship for the first time in Bangladeshi patients.Although our study had low power (70%), we report here that the TgAb and TPOAb tests have a modest level of diagnostic accuracy.
FNAC is the investigation of choice for diagnosing malignant thyroid nodules.The measurement of thyroid autoantibodies would contribute to a better diagnosis of carcinoma differentiation in thyroid nodules than the FNAC. 16Although small, there is a chance of spreading the underlying cancer by the FNAC procedure.
Scientists have already claimed that serum thyroid autoantibody levels as biochemical markers would provide an opportunity for better management planning. 16i et al. mentioned a higher prevalence of malignancy (18.8%) in individuals with elevated TPOAb levels compared to those (9.2%) with normal TPOAb levels. 17  explanation might be the local inflammatory reaction of tumour cells with the production of TPOAb. 18Our findings were similar to the findings of the above reports.
In the current study, TgAb levels significantly differed between malignant and benign thyroid nodules.74.4% and 70.4%, respectively. 19This might be due to the differences in study design and participant background.
Human thyroglobulins have four to six epitopes that are recognised by B cells. 20Lupoli et al. recently determined the prognostic relevance of TgAb epitope specificities in thyroid cancer. 21Therefore, it is possible that occult thyroid cancer stimulates chronic immunologic responses and produces new or more TgAb.This could explain the mechanism of association between positive TgAb and malignancy.

Conclusion
Although the study findings are based on a small sample, our findings indicate that thyroid cancer is associated with high TgAb and TPOAb levels.They have a modest accuracy in detecting malignant thyroid nodules.Therefore, elevated levels of TgAb and TPOAb may be considered candidates for screening for nodular malignancy in the thyroid.
minutes.The separated serum specimen was kept in an Eppendorf tube and stored at -20º C until analysis was performed.Laboratory investigations were done at the Department of Microbiology and Immunology, BSMMU.All samples were tested on four successive occasions within one month of sample collection.

TABLE 2 Logistic regression analysis for predicting factors thyroid malignancy
a Confidance interval

TABLE 1 Mean (standard deviation) of age and antibodies in malig- nant and benign thyroidnodule groups (n=104)
a Thyroglobulin antibody, b Thyroid peroxidase antibody